tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation
tBid elicits a conformational alteration in membrane-bound Bcl-2 such that it inhibits Bax pore formation
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DOI：
10.1074/jbc.m608303200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Lin, Jialing
中科院分区：
文献类型：
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作者：
Peng, Jun;Tan, Chibing;Lin, Jialing
在细胞凋亡的启动过程中，Bcl-2家族蛋白调节线粒体外膜的通透性。仅BH 3蛋白tBid激活促凋亡Bax以从线粒体释放细胞色素c。tBid还激活线粒体外膜中的抗凋亡Bcl-2，将其从单跨构象改变为多跨构象，其结合活性Bax并抑制细胞色素c释放。然而，目前尚不清楚是否需要其他线粒体蛋白引起tBid诱导的Bcl-2构象改变。为了确定功能上重要的Bcl-2构象改变所需的最小组分，我们使用纯化的蛋白质和脂质体重建反应。我们发现纯化的tBID足以诱导脂质体束缚的Bcl-2的构象改变，但不能诱导胞质中的Bcl-2的构象改变，从而产生与药物处理细胞线粒体外膜中发现的多跨形式相似的多跨形式。取消tBid/Bcl-2相互作用的突变也取消了构象改变，表明在膜上的直接tBid/Bcl-2相互作用是必需的，并且足以引起构象改变。此外，活性Bax还引起Bcl-2构象的改变。Bcl-2突变体在构象改变试验中显示出增加或降低的活性，在体外抑制Bax孔形成和在体内防止细胞凋亡中显示出相应的活性。因此，在体外和体内，膜结合的Bcl-2和tBid与Bcl-2的活化的直接相互作用之间存在强相关性。
During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid,activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multispanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multispanning form that is similar to the one found in the mitochondrial outer membrane of drug-treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay showed corresponding activities in inhibiting pore formation by Bax in vitro and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.